This application outlines studies to further explore the process of HIV-1 integration and integrase function. The Applicant has developed a synoptic set of 82 charged cluster-to-alanine mutants of integrase covering 93% of the charged residues, and inserted these into an infectious molecular clone of HIV-1. He proposes to examine several aspects of integrase biology in 3 Specific Aims. Aim 1 will utilize a panel of 5 conditional integrase mutants to recover intra- and extragenic viral suppressor mutants that suppress the original mutant phenotype. It is hoped that this will lead to identification of viral ligands that make critical intermolecular contact with the integrase protein during virus replication. An HIV shuttle vector system has been developed which allows direct cloning of circular DNA forms into bacteria, and which will facilitate recovery and characterization of these suppressor mutants. Specific Aim 2 will characterize the limited gene expression seen from circular DNA forms derived from certain replication-defective integrase mutants. Kinetics, copy number, and protein expression in primary cells will be assessed using real-time PCR and reporter assays. These studies may have relevance to gene therapy protocols and vaccine development. Aim 3 will further analyze a newly identified potent nuclear localization signal present in the carboxy terminus of integrase. Mutations in this region will be used to assess the role of this NLS in infection of non-dividing cells.